CN112142849B - Short peptide inhibitor targeting calmodulin phosphatase and substrate T cell activated nuclear factor thereof and application thereof - Google Patents

Abstract

The invention discloses a short peptide inhibitor targeting calmodulin phosphatase and a T cell activating nuclear factor as a substrate thereof and application thereof. The invention provides a fusion polypeptide, which is formed by connecting EV motif of calmodulin phosphatase regulatory factor 1 and LxVP motif of T cell activation nuclear factor 1 through A238L connecting peptide, or is obtained by connecting membrane-penetrating peptide at amino terminal and/or carboxyl terminal. The pep3 short peptide constructed by the invention has stronger capacity of being combined with CN and inhibiting enzyme activity, plays an immunosuppressive role in an intracellular CN/NFAT signal path, can more specifically destroy the CN/NFAT interaction than the traditional inhibitor environmental protection rhzomorph A, has an immunosuppressive function at an animal level, and can be used as a tool medicine for developing local immunosuppression or other immunosuppressions.

Description

Short peptide inhibitor targeting calmodulin phosphatase and substrate T cell activated nuclear factor thereof and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a short peptide inhibitor targeting calmodulin phosphatase and a T cell activating nuclear factor as a substrate thereof and application thereof.

Background

Calmodulin phosphatase (CN) is Ca-dependent²⁺And calmodulin (CaM) serine/threonine protein phosphatases that interact with substrate and target proteins in the organism. CN is a different sourceDimer, consisting of catalytic subunits (A subbunit, CNA) and regulatory subunits (B subbunit, CNB). The two subunits are tightly bound and only become separated by denaturation. Only in the presence of CNA, its activity is very low, and only in the presence of CNB, a highly specific phosphatase activity is exhibited. When Ca is present²⁺Binding to CNB has a major effect on the activity of CN, but the more major effect is due to the modulation of CaM.

The activation of nuclear factor of activated T-cells (NFAT) is one of the important substrates of CN, and the activation depends on the dephosphorylation of CN, and the CN/NFAT signal path participates in important physiological and pathological processes of immune response, nervous system development, learning and memory formation in vivo. NFAT has two motifs (motifs) that bind CN, namely pxijit motif and LxVP motif. The classical immunosuppressive drug cyclosporin A (CsA) acts as an immunosuppressive agent by competing with LxVP motif of NFAT to disrupt CN/NFAT interaction.

The classical immunosuppressant, cyclosporin A, (cyclosporine A) requires binding to the intracellular cyclosporin Cyclophilin in order to exert a dephosphorylation effect on CN. It has many problematic toxic and side effects, and the main adverse reactions are nephrotoxicity, neurotoxicity and hepatotoxicity, which may cause long-term failure of transplanted and normal kidneys. It also aggravates hypertension and hyperlipidemia, thus causing cardiovascular disorders and increasing the risk of obesity.

Calmodulin phosphatase regulatory factors (RCANs) are endogenous regulatory proteins of CN and regulate the CN/NFAT signaling pathway in vivo by affecting the activity of CN, and its binding to CN is also dependent on its PxIxIT motif and LxVP motif.

Disclosure of Invention

The invention aims to provide a short peptide inhibitor targeting calmodulin phosphatase and a T cell activating nuclear factor as a substrate thereof and application thereof.

In a first aspect, the invention claims a fusion polypeptide.

The fusion polypeptide claimed by the invention is (A1) or (A2):

(A1) thefusion polypeptide 1 is formed by connecting an EV motif (EV motif) of calmodulin phosphatase regulatory factor 1(RCAN1) and an LxVP motif (LxVP motif) of T cell activating nuclear factor 1(NFATc1) through an A238L connecting peptide;

(A2) and (b)fusion polypeptide 2, which is obtained after connecting a membrane-penetrating peptide to the amino terminal and/or the carboxyl terminal of the fusion polypeptide 1 (A1).

Wherein the EV motif of the calmodulin phosphataseregulatory factor 1 is a polypeptide shown in SEQ ID No. 1. The LxVP motif of the T cell activationnuclear factor 1 is polypeptide shown as SEQ ID No. 2. The A238L connecting peptide is polypeptide shown in SEQ ID No. 3. The cell-penetrating peptide is a polypeptide shown in 1 st to 14 th positions of SEQ ID No. 5.

Further, thefusion polypeptide 1 is a polypeptide shown as SEQ ID No.4 (named pep 3). Thefusion polypeptide 2 is a polypeptide shown as SEQ ID No.5 (named as 11R-pep 3).

In a second aspect, the invention claims a modified fusion polypeptide.

The modified fusion polypeptide of the present invention is obtained by performing fluorescence labeling on the amino terminal and/or the carboxyl terminal of the fusion polypeptide of the first aspect.

Wherein, the fluorescent label can be any fluorescent label capable of labeling the polypeptide.

In a specific embodiment of the present invention, the modified fusion polypeptide is specifically any one of the following:

(B1) the amino terminal of thefusion polypeptide 1 is fluorescently labeled by FAM or FITC;

(B2) the amino terminal of thefusion polypeptide 2 is labeled by FITC fluorescence.

In a third aspect, the invention claims nucleic acid molecules encoding the fusion polypeptides described hereinbefore.

Further, in the nucleic acid molecule, the nucleic acid molecule encoding the EV motif of calmodulin phosphataseregulatory factor 1 is a DNA molecule represented by SEQ ID No. 7. The nucleic acid molecule of the LxVP motif of the T cell activationnuclear factor 1 is a DNA molecule shown in SEQ ID No. 8. The nucleic acid molecule for coding the A238L connecting peptide is a DNA molecule shown in SEQ ID No. 9.

Furthermore, the nucleic acid molecule encoding thefusion polypeptide 1 is a DNA molecule shown in SEQ ID No. 10.

In a fourth aspect, the invention claims recombinant vectors, expression cassettes, recombinant bacteria or transgenic cell lines containing said nucleic acid molecules.

Wherein the expression cassette comprises a promoter, the nucleic acid molecule (encoding gene) and a termination sequence in this order from upstream to downstream. The recombinant vector may be a recombinant vector containing the expression cassette. The recombinant strain can be obtained by introducing the recombinant vector into a recipient strain. The transgenic cell line can be obtained after introducing the recombinant vector into a receptor cell.

In a fifth aspect, the invention claims any of the following applications:

(C1) the use of a fusion polypeptide or a modification of said fusion polypeptide or of said nucleic acid molecule or of said recombinant vector or of said expression cassette or of said recombinant bacterium or of said transgenic cell line as hereinbefore described for the preparation of an immunosuppressant;

(C2) use of the fusion polypeptide or the fusion polypeptide modification or the nucleic acid molecule or the recombinant vector or the expression cassette or the recombinant bacterium or the transgenic cell line as hereinbefore described for the preparation of a product for the prevention and/or treatment of hypersensitivity diseases;

(C3) use of the fusion polypeptide or the fusion polypeptide modification or the nucleic acid molecule or the recombinant vector or the expression cassette or the recombinant bacterium or the transgenic cell line as described hereinbefore for the preparation of a product for the prevention and/or treatment of an autoimmune disease;

(C4) use of the fusion polypeptide or the fusion polypeptide modification or the nucleic acid molecule or the recombinant vector or the expression cassette or the recombinant bacterium or the transgenic cell line as described hereinbefore for the preparation of a product for the prevention and/or treatment of allergy;

(C5) use of the fusion polypeptide or the fusion polypeptide modification or the nucleic acid molecule or the recombinant vector or the expression cassette or the recombinant bacterium or the transgenic cell line as described hereinbefore for the preparation of a product for the prevention and/or treatment of asthma;

(C6) use of a modified fusion polypeptide according to any one ofclaims 1 to 3 or a modified fusion polypeptide according toclaim 4 or a nucleic acid molecule according to any one ofclaims 5 to 7 or a recombinant vector, expression cassette, recombinant bacterium or transgenic cell line according toclaim 8 for the manufacture of a product for inhibiting immune rejection in an organ transplant.

In a sixth aspect, the invention claims the use of the fusion polypeptide or the modified fusion polypeptide or the nucleic acid molecule or the recombinant vector or the expression cassette or the recombinant bacterium or the transgenic cell line as described above for the preparation of a product having at least one of the following functions:

(D1) binding to calmodulin phosphatase (CN);

(D2) inhibiting dephosphorylation of a substrate by calcineurin;

further, the substrate is a RII peptide;

furthermore, the RII peptide is a polypeptide shown as SEQ ID No. 6;

(D3) the nuclear entry behavior of the T cell activated Nuclear Factor (NFAT) that inhibits Ionomycin (Ionomycin) activation;

(D4) inhibiting dephosphorylation of a T cell activating nuclear factor by calcineurin phosphatase;

(D5) inhibiting expression of a downstream gene that is initiated by a T cell activating nuclear factor;

further, the downstream gene is IL-2 and/or TNF-alpha. The expression may be at the transcriptional level or at the protein level.

Compared with CsA, the pep3 short peptide constructed by the invention can simultaneously occupy two action sites for combining CN and NFAT, has stronger activity for CN combining ability and enzyme activity inhibition, plays an immunosuppressive role in an intracellular CN/NFAT signal pathway, can more specifically destroy CN/NFAT interaction than the traditional inhibitor environmental protection bacterin A, and has an immunosuppressive function at an animal level. Can be used as a tool medicine for local immunosuppression development or for development of other immunosuppression.

Drawings

FIG. 1 shows design concept and sequence of pep3 short peptide.

FIG. 2 shows the comparison of the binding capacity of pep3 short peptide to pep1 and pep2 short peptides ("calmodulin phosphatase A subunit" is the amount of calmodulin phosphatase A subunit pulled down by each group of short peptides, "upper calmodulin phosphatase A subunit" is the amount of fresh rat brain lysate added to each group of short peptides, "glutathione mercaptotransferase fusion protein" is the equivalent expression level of each group of short peptides, and the statistical results of the gray scale analysis of CNA bands are shown on the right.^**P<0.05,^***P<0.001；n＝3)。

FIG. 3 shows the inhibition of CN activity by pep3 short peptide.

FIG. 4 shows the dissociation constants of pep3 for short peptide and CN (left is the dissociation curve of pep3 from CN high affinity site, right is the dissociation curve of pep3 from CN low affinity site).

FIG. 5 shows the construction and identification of a plasmid for eukaryotic expression of pep3 gene. a is the result of pep3 PCR; b is the enzyme digestion result of Setd 3-Flag-pcDNA3.0; c, positive clone PCR verification; d is the result of enzyme digestion verification of the positive clone.

FIG. 6 shows the intracellular binding capacity of pep3 short peptide to CN.

FIG. 7 shows the effect of pep3 short peptide on NFAT nuclear entry behavior.

FIG. 8 shows the effect of pep3 on the dephosphorylation of NFAT protein (with cyclosporin as a positive control drug, compared to the control group, it can be seen that pep3 can effectively inhibit the dephosphorylation of NFAT by CN and confine it to the cytoplasm, blocking its activation into the nucleus).

FIG. 9 shows the effect of pep3 on IL-2 transcript levels.

FIG. 10 is a graph showing the effect of pep3 short peptide on the level of TNF-. alpha.transcription.

FIG. 11 shows the entrance of pep3 short peptide modified by transmembrane sequence (cells incubated with FITC-11R-pep3 transmembrane short peptides at different concentrations of 1. mu.M, 2. mu.M, 5. mu.M and 10. mu.M).

FIG. 12 shows the effect of the transmembrane sequence modified pep3 short peptide on the dephosphorylation of endogenous NFAT protein (from left to right:Lane 1 is the untreated control,Lane 2 is the transfected cells with 2. mu.M cyclosporin A ion as a positive control drug,Lane 4 is the pep3 short peptide inhibition with 1. mu.M incubation,Lane 5 is the pep3 short peptide inhibition with 5. mu.M incubation,Lane 3 is the blank control with ionomycin stimulation but no inhibitor added).

FIG. 13 shows the effect of a pep3 short peptide modified with a transmembrane sequence on the transcriptional level of the IL-2 gene.

FIG. 14 shows the effect of pep3 short peptide modified by transmembrane sequence on the protein expression level of IL-2 gene.

FIG. 15 is a graph of the effect of a pep3 short peptide modified with a transmembrane sequence on the development of asthma in mice.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 design of pep3 short peptide targeting calmodulin phosphatase and its substrate T cell activated nuclear factor and Synthesis of Gene sequence

The invention relates to an active polypeptide which has two binding sites with CN and is named peptide3 (pep3 for short) by connecting EV motif (pep1) of RCAN1 and LxVP motif (pep2) of NFATc1 by using 17 amino acids of short peptide A238L as a Linker between PxIxIT site and LxVP site of CN and a substrate thereof (figure 1). The amino acid sequences of the three pep short peptides and Linker connecting peptide are shown in Table 1.

TABLE 1 amino acid sequence of each short peptide of the present invention

According to the codon bias of mammals, nucleic acid sequences were synthesized in Huada Gene biotechnology, Inc. according to the amino acid sequence shown in FIG. 1. The nucleic acid sequence of the Pep1 short peptide is shown in SEQ ID No. 7. The nucleic acid sequence of the Pep2 short peptide is shown in SEQ ID No. 8. The nucleic acid sequence of the coding Linker (A238L short peptide) is shown in SEQ ID No. 9. The nucleic acid sequence of the Pep3 short peptide is shown in SEQ ID No. 10.

Example 2, the binding ability of pep3 short peptide to CN is much stronger than that of pep1 and pep 2-GST Pull-Down experiment

1. Construction of recombinant expression vectors

The optimized Pep1, Pep2 and Pep3 short peptide coding genes in theembodiment 1 are respectively cloned to the multiple cloning sites of the pGEX-4T-1 vector for fusion expression of GST tag protein, so that the short peptide and the GST tag protein can be fusion expressed, and three recombinant expression vectors are obtained.

The recombinant expression vector for expressing the Pep1 short peptide is named as pGEX-4T-1-Pep1 after the sequencing verification is correct; the recombinant expression vector for expressing the Pep2 short peptide is named as pGEX-4T-1-Pep2 after the sequencing verification is correct; the recombinant expression vector for expressing the Pep3 short peptide is named pGEX-4T-1-Pep3 after the sequencing verification.

2. Transformation of Escherichia coli

The three recombinant expression vectors are respectively introduced into an escherichia coli receptor competent cell, and the specific operation is as follows:

(1) 50 mu l of E.coli competent cell DH5 alpha is taken to be iced and melted for 5min, 2.5 to 5 mu l of recombinant expression vector (less than or equal to 10ng) constructed in thestep 1 is added and mixed evenly, and the mixture is kept stand on ice for 30 min;

(2) thermally exciting for 60-90 s in water bath at 42 ℃;

(3) quickly placing on ice for cooling for 2-3min after heat shock;

(4) adding 800 μ l of LB liquid culture medium without antibiotic into the tube, mixing gently, and placing at 37 ℃ x 160rpm for shaking culture for 1 h;

(5) centrifuging at room temperature multiplied by 4,000rpm multiplied by 1-3 min;

(6) abandoning 700 mul of culture medium, then re-suspending the thalli, taking 100 mul of bacterial liquid, coating the bacterial liquid on the LBA solid culture medium, and placing the bacterial liquid for 30min with the front side upward;

(7) and (4) inverting the culture dish after the bacterial liquid is completely absorbed by the culture medium, and performing inverted culture at 37 ℃ for 12-16 h.

(8) Selecting a single clone, inoculating the single clone into 20ml of liquid LBA culture medium, and carrying out shaking culture at 37 ℃ and 190rpm for 12-14 h;

(9) and (3) amplification culture: adding the resuscitated culture solution into 500ml of TM culture medium (Tryptone 12 g/L; Yeast Extraction 24 g/L; NaCl 10 g/L; Glycerin 6ml/L), performing shake culture at 37 ℃ and 250rpm for about 3h, and reserving a small amount of fresh TM culture medium (OD value is average +0.1/10min) during inoculation;

(10) inducing expression: culturing until OD is measured₆₀₀When the concentration is 0.6-0.8, IPTG is added into the culture medium to the final concentration of 100 mu M, and the culture is carried out for 14h under the condition of shaking at 25 ℃ and 190 rpm;

(11) collecting bacteria: centrifuging at 4 deg.C and 5000rpm for 20min, discarding supernatant, and freezing at-20 deg.C.

3. Thallus lysis (crude extraction of pep short peptide)

Weighing a 2ml EP tube, deducting and weighing thalli with the size of a small finger cap, weighing, adding 10ml/g of thallus homogenate (7 Xprotease inhibitor is added into precooled TBS to 1X; PMSF to 0.1 mM; DTT to 2 mM; beta-ME 0.1% (V/V)) and mixing evenly, preparing a beaker, ice, a ruler and carrying out ultrasonic crushing; centrifuging at 4 ℃ for 13000rpm for 30 min; the supernatant protein solution was taken in a new 1.5ml EP tube and the protein concentration was measured by the Bradford method.

4. Media processing

Taking 50 mul of Sepharose 4B medium mixed uniformly by an inlet gun head, centrifuging in a 1.5ml EP tube at 4 ℃ multiplied by 13000rpm multiplied by 1min, and carefully discarding supernatant; add 500. mu.l Wash Buffer (TBS solution, THIS-HCL solution with pH 8.0) per tube and blow-pipette the resuspension medium, centrifuge at 4 ℃ X13000 rpm X1 min,repeat 5 times; adding 500 mul of thallus lysis protein liquid into each tube of medium, placing on a vertical suspension instrument at 4 ℃, and slowly rotating for 3 h; centrifuging at 4 ℃ for 13000rpm for 1min, and carefully discarding the supernatant; add 500. mu.l of Wash Buffer slowly along the tube wall (same above), mix the Wash medium gently by inversion, centrifuge at 4 ℃ X13000 rpm X1 min, discard the supernatant carefully andrepeat 5 times.

5. Rat brain lysis

Preparing precooled normal saline, alcohol, sterilized scissors, forceps and medical cotton, using a disposable 1ml injector, and weighing a 2ml EP tube; killing one Balb/c mouse by a neck-breaking method, dipping cotton into alcohol to wipe the head of the mouse, shearing the skin of the mouse, pushing a scissors to the joint of a skull to cut, taking the mouse brain into precooled physiological saline by using a pair of tweezers, washing twice, dividing the whole brain into 1/4-1/2 sizes, subpackaging the whole brain in a 2ml EP tube, weighing and storing in a refrigerator at the temperature of-20 ℃; taking one part, adding 150-200 mu l/20mg of tissue lysate, homogenizing by using a 1ml injector to a degree that the tissue lysate can be blown and sucked by using a needle head, and carrying out ultrasonic crushing; centrifuging at 4 ℃ for 13000rpm for 60 min; the supernatant was pipetted into a clean EP tube and its concentration was measured by the Bradford method.

6. Pull Down experiment

Adding 500 μ l of the mouse brain lysis protein solution obtained instep 5 into each tube of medium (obtained in step 4), placing on a 4 deg.C vertical suspension apparatus, and slowly rotating for 1 h; centrifuging at 4 ℃ for 13000rpm for 1min, and carefully discarding the supernatant; slowly adding 500. mu.l of Wash Buffer (same as above) along the tube wall, gently inverting and mixing the cleaning medium, centrifuging at 4 ℃ for 13000rpm for 1min, carefully discarding the supernatant, and repeating for 7 times; add 20. mu.l of 2 × protein loading Buffer to each tube of medium, boil in water bath for 5min, and store in a refrigerator at-20 ℃.

7、Western Blot

(1) Centrifuging the sample treated in thestep 6 at 4 ℃ multiplied by 10000rpm multiplied by 5 min; preparing SDS-PAGE gel: cleaning the rubber plate and checking leakage, pouring 12% of separation rubber into the rubber plate until the separation rubber is 3-5 cm away from the short plate, and slowly sealing with ethanol; standing for about 30min until the separation gel is solidified, adding 4% concentrated gel along the wall, inserting a comb, and standing for solidification. Electrophoresis: the gel plate was placed, the electrophoresis apparatus was connected, and 30. mu.l of gel was applied to each well to start electrophoresis (80V 30 min; 120V 90 min).

(2) Film transfer: precooling 1 × blowing Buffer, and preparing anhydrous methanol; shearing a PVDF membrane according to the size of the gel after electrophoresis, and activating for 1min by using anhydrous methanol; starting to rotate the film: (150 mA-250 mA 120min)

(3) And (3) sealing: preparing 5% (m/V) skimmed milk powder solution by using 1 × TBST Buffer, and slowly incubating for 1h at room temperature; primary antibody hybridization: a5% (m/V) skim milk powder solution was prepared using 1 XTSSBuffer, according to the following 1: 2000-1: GST monoclonal antibody was diluted with 5000, and incubated at 4 ℃ overnight slowly; rinsing: rapidly washing with 1 × TBST for 5min, and repeating for 3 times; (recovery of primary antibody) secondary antibody hybridization: a5% (m/V) skim milk powder solution was prepared using 1 XTSSBuffer, according to the following 1: 5000-1: 10000 to dilute the Goat Anti-mouse IgG, and slowly incubate for 1h at room temperature; rinsing: rapidly washing with 1 × TBST for 5min, and repeating for 3 times; and (5) developing and exposing.

8. Results

By using GST Pull-down experiments and comparing the binding capacity of three short peptides pep1, pep2 and pep3 to CN in mouse brain in vitro, the binding capacity of pep3 to CN is obviously stronger than that of pep1 and pep2 (figure 2).

Example 3 inhibition of CN Activity by pep3 short peptide-Malachite Green assay Using RII short peptide as substrate

The pep3 short peptide used in the present embodiment was synthesized by Zhongke Asia photo biology, and its amino acid sequence is shown in SEQ ID No. 4.

1. Proenzyme activity assay

The proenzyme activity of the catalytic subunit of CN (catalytic subunit, A subunit, CNA) on the CN physiological substrate RII short peptide (RII subunit of protein phosphokinase, amino acid sequence is shown in Table 1) was determined. The experimental methods are shown in table 2.

TABLE 2 protocol for determination of CNA proenzyme Activity

Note: the enzyme solution in the table is CNA proenzyme solution (enzyme solution with higher CN concentration obtained by splitting rat brain). The enzyme diluent was a diluent for diluting the CNA proenzyme solution (formulation: 50mM (pH 7.4) Tris-HCl; 0.1mM EDTA; 0.1mM EGTA; 0.2% (v/v) NP-40; 1mM (currently used plus) DTT; 1mM (currently used plus) PMSF; 1X (currently used plus) protease inhibitor). The concentration of the RII solution was 0.75 mM.

2. Determining the influence of pep3 on the CNA enzyme activity

Appropriate CNA concentrations were diluted according to the proenzyme activity measured instep 1. The experiment was then carried out according to the protocol shown in table 3.

TABLE 3 protocol for determining the Effect of pep3 short peptide on CNA enzyme Activity

Note: the enzyme solution, enzyme diluent and RII solution are shown in Table 2. The peptide solution is pep3 short peptide solution for test, and the concentration gradient is in series; the peptide solution is used for dissolving pep3 short peptide, specifically ddH₂O。

The relative enzyme activity of CN added with pep3 short peptide solution with series concentration gradient is calculated according to the following method:

the enzyme activity calculation method comprises the following steps:

definition of enzyme activity unit (U): the pH was 7.4 and the temperature was 30 ℃ and the amount of enzyme required to release 1nmol of product per minute of reaction was one activity unit.

"enzyme specific activity" is uniformly used to represent the activity state of the enzyme, and is defined as: a pH of 7.4, a temperature of 30 ℃ and the amount of substance of substrate (RII) that is catalytically hydrolyzed per unit weight of enzyme per unit time, in units of: pmol/(min. mg).

The specific calculation method is as follows:

(1) measuring the change of OD value generated by CN dephosphorization;

(2) will OD₆₂₀(CN) conversion to free phosphate ion increment: will OD₆₂₀(CN) was substituted into the fitted formula, and the amount of the phosphoric acid substance was evaluated. The method for obtaining the fitting equation is shown as the following "calibration of phosphoric acid standard curve";

(3) calculating the specific Activity of CN

U (CN) ═ phosphate ion increment/(amount of protein per well. times enzyme reaction time)

Relative enzyme activity ═ (peptide test group u (cn) -peptide control group u (cn))/(enzyme test group u (cn) -enzyme control group u (cn) -x 100%.

Calibration of phosphoric acid standard curve:

1) 1ml of 1 XCA was prepared²⁺Activity buffer (using 500. mu.l ddH)₂O dilution of 500. mu.l of 2 XCA²⁺Detect live buffer). Wherein, 2 XCA²⁺The measured activity buffer formula is shown in Table 4.

TABLE 42 XCA²⁺Formula of activity buffer

2) Phosphate standards (80. mu.M) were diluted in duplicate and a series of concentration gradient reaction wells were prepared in 96-well plates. Phosphoric acid standard solutions at concentrations of 40, 20, 10, 5, 2.5, 1.25 and 0.625. mu.M contained 2, 1, 0.5, 0.25, 0.125, 0.063 and 0.031nmol PO, respectively₄^3-. 2nmol PO was added to both A1 and B1 wells₄^3-(ii) a 1nmol PO was added to both B2 and A2 wells₄^3-(ii) a 0.5nmol PO was added to both C1 and C2 wells₄^3-(ii) a 0.25nmol PO was added to both D1 and D2 wells₄^3-(ii) a 0.125nmol PO was added to both E1 and E2 wells₄^3-(ii) a 0.063nmol PO was added to both F1 and F2 wells₄^3-(ii) a 0.031nmol PO was added to both G1 and G2 wells₄^3-(ii) a 0nmol PO was added to both H1 and H2 wells₄^3-. The specific operation is as follows:

A) 50. mu.l of 2 XCA were added to each of wells A1 and A2²⁺Testing a live buffer;

B) 50. mu.l of 1 XCA was added to each of wells B1-H1 and B2-H2²⁺Testing a live buffer;

C) respectively adding 50 mul of phosphoric acid standard substance with the concentration of 80 mu M into the holes A1 and A2, and repeatedly blowing and uniformly mixing by using a pipette gun;

D) sucking 50 mul of mixed solution from the hole A1, transferring the mixed solution into the hole B1, and repeatedly blowing and uniformly mixing the mixed solution by using a liquid transferring gun;

E) sucking 50 mul of mixed solution from the hole B1, transferring the mixed solution into the hole C1, and repeatedly blowing and uniformly mixing the mixed solution by using a liquid transferring gun;

F) after mixing, the above steps were repeated until wells D1-G1, and 50. mu.l of the mixture was discarded from well G1, taking care not to perform this operation on well H1, and well H1 was blank zero-calibration. All wells were 50 μ l in volume.

G) The same procedure was used for wells A2-G2.

3) Adding 100 μ l malachite green reagent into each well, reacting at room temperature for 20-30min, and reading OD with microplate reader₆₂₀Numerical value in PO₄^3-Concentration is in the abscissa, OD₆₂₀The value is the ordinate, a phosphoric acid standard curve is drawn, and dephosphorizing acid is obtainedCalculating the formula: 0.1577x +0.0682 (R)²＝0.9808)。

3. Results

The activity of pep3 on dephosphorylation of a protein substrate by CN was determined using a physiological substrate RII short peptide of CN (RII subunit of protein phosphokinase), and the result showed that pep3 short peptide inhibits half of CN at 400nM (FIG. 3).

Example 4 determination of dissociation constant of pep3 short peptide from CN-micro thermal surge

The N-terminus of pep3 short peptide used in this example was fluorescently labeled with FAM (5-carboxyfluorescein, 5-Carboxy-fluorescein). Synthesized by Zhongke matt Bio Inc.

The concentration of the catalytic subunit of CN (A subbunit, CNA) was 70. mu.M, and equimolar amounts of the regulatory subunit of CN (CNB) and calmodulin (CaM) were added, together with 1.5. mu.M Ca²⁺Finally, the CNA mixture was diluted to 50. mu.M (where 50. mu.M means that the CNA/CNB/CaM concentration in the mixture was 50. mu.M), and stored at 80 ℃. Measuring the initial absorbance (. ltoreq.200 counts) of peptide dilution Buffer (50mM (pH 7.4, 4 ℃ C.) Tris-HCl) and CNA mixture dilution Buffer (formulation: 50mM (pH 7.4) Tris-HCl; 1mM PMSF) (excluding the effect of fluorescence generated by non-fluorescent peptides on the experimental results); fluorescent FAM-pep3 short peptide was dissolved in DMSO to a stock concentration of 1mM, and FAM-pep3 short peptide at an initial concentration of 500nM was diluted in duplicate in a brown shaded EP tube.

Ultraviolet absorbance of each concentration of FAM-pep3 short peptide is detected, and FAM-pep3 short peptide with the concentration of 62.5nM is selected as a proper working concentration. Taking 16 special EP tubes for MST, adding 10 mu l CNA mixed solution into each tube to dilute Buffer, and taking the CNA mixed solution with the initial concentration of 50 mu M to carry out multiple dilution; to the diluted CNA mixture was added 10. mu.l of 62.5nM FAM-pep3 short peptide solution, mixed well and allowed to stand at room temperature for 5 min.

3. And (3) taking a K002 type capillary tube to dip the mixed solution, sequentially placing the mixed solution on a tube frame, and measuring the mixed solution by a well-managed cabin door.

4. Results

Using a microcalorimetric surge experiment (MST), pep3 with a fluorescent label of FAM (5-carboxyfluorescein, 5-Carboxy-fluorescein) was determined to have two binding sites with a high affinity site dissociation constant of about 8. + -. 1.2nM and a low affinity site dissociation constant of about 1.60. + -. 0.20. mu.M with CN (FIG. 4).

Example 5 binding ability of pep3 short peptide to intracellular CN-Co-IP experiment

1. Construction of recombinant expression vectors

The coding gene of the Pep3 short peptide optimized according to the codon preference of the mammal in theembodiment 1 is cloned between the enzyme cutting sites Xho I and Sac I of the eukaryotic expression plasmid Setd3-Flag-pcDNA3.0(addgene, cat # 49144) for fusion expression of the Flag tag protein, so that the short peptide and the Flag tag protein can be subjected to fusion expression to obtain the recombinant expression vector. The specific operation is as follows:

pGEX-4T-1-Pep3 is used as a template, a Primer (Primer-F: CCGCTCGAGATGCCAGGAGAGAAGTATG; Primer-R: CAGGAGCTCTTACTTCGCCCACTGGTAG) is designed to carry out PCR reaction, the Pep3 gene is amplified, as shown in a in figure 5, the vector Setd3-Flag-pcDNA3.0 and a PCR product are subjected to enzyme digestion and recovery, as shown in b in figure 5, T4 ligase is used for connection, the vector is transformed into DH5a escherichia coli to carry out positive cloning identification, bacterial liquid PCR verification and enzyme digestion verification are carried out, as shown in c and d in figure 5, sequencing of the warble gene proves that the recombinant expression vector is successfully constructed.

The recombinant expression vector for expressing the Pep3 short peptide was designated Pep3-Flag and verified to be correct by sequencing.

2. Transfection of HeLa cells

Mu.g of the recombinant expression vector pep3-Flag described above was transfected into Hela cells.

3. Treatment Medium + Loading

Taking 50 μ l of Protein A/G plus Agarose medium mixed uniformly by an inlet gun head, centrifuging at 4 ℃ multiplied by 5000rpm multiplied by 1min in a 1.5ml EP tube, and carefully discarding supernatant; adding 500 μ l Wash Buffer I (i.e. 1 × TBS) into each tube, blowing and beating the heavy suspension medium, centrifuging at 4 ℃ for 5000rpm for 1min, and repeating for 3 times; 200 mul Wash Buffer I (same as above) was added to each tube of medium, and then Flag monoclonal antibody (mouse)/CNA monoclonal antibody (rabbit) was added according to the antibody specification (1: 50), and normal IgG of the same species (i.e. other mouse monoclonal antibodies/rabbit monoclonal antibodies) was added to the control group, and the mixture was placed in a vertical mixer and suspended at 4 ℃ for 2 hours.

4. Collecting cells

Gently shaking the culture dish, washing the semi-floating cells, and discarding the culture medium; 1ml of precooled PBS was added to rinse the medium; discarding PBS, adding 1ml pancreatin, digesting for about 1min, blowing cells into 1.5ml EP tube, centrifuging at 4 deg.C for 1000rpm for 5 min; the supernatant was discarded and 100. mu.l/10 of the supernatant was added⁶Uniformly mixing cell lysate of each cell, suspending, standing in ice bath for 30min, and vortexing once every 5 min; centrifuging at 4 ℃ for 13000rpm for 10 min; collecting supernatant, and measuring the protein concentration by Bradford method (leaving 20 μ l of sample per piece of gel as Input, adding 20 μ l of 2 xSDS-loading Buffer, mixing, boiling for 5min, and storing at-20 deg.C); the protein solution was diluted to consistency and CaCl was added to the protein solution to a final concentration of 1mM₂And the final concentration is 2 MuM CaM, and the mixture is uniformly mixed for standby.

5、Co-IP

Centrifuging the medium (treated instep 3, when the medium has bound the antibody) at 4 ℃ X5000 rpm X1 min, carefully discarding the supernatant; slowly adding 500 μ l of Wash Buffer I along the tube wall (same as step 3), gently reversing and uniformly mixing the cleaning medium, centrifuging at 4 ℃ multiplied by 5000rpm multiplied by 1min, carefully discarding the supernatant, leaving a small part of the supernatant, standing on ice for later use, and repeating for 3 times; diluting the protein solution (obtained in the step 4) into uniform protein mixed solution, adding the uniform protein mixed solution into a medium, placing the medium on a vertical mixer, and slowly rotating for 3 hours at the temperature of 4 ℃; centrifuging at 4 deg.C for 5000rpm for 1min, and removing supernatant; slowly adding 500 μ l of Wash Buffer II (also 1 × TBS) along the tube wall, gently inverting and mixing the cleaning medium, centrifuging at 4 deg.C × 5000rpm × 1min, and repeating for 3 times; adding 20 μ l of 2 × loading buffer, boiling in water bath for 5min, and freezing to-20 deg.C in refrigerator for use.

Western Blot was then carried out in accordance with step 7 of example 2.

6. Results

Co-immunoprecipitation (Co-IP) experiments were performed on cell lysates and found that pep3 short peptide was still able to bind CN under physiological conditions (FIG. 6).

Example 6 inhibition of ionomycin-activated NFAT Nuclear entry behavior by pep3 short peptide-immunofluorescence

1. Cell processing

(1) The density of Hela cells in the fast growth phase of the inoculation is 5 multiplied by 10⁵Placing the cells in a 24-well plate with a cell slide, and culturing for 16-18 h (less than or equal to 24h) in a cell culture box at 37 ℃, wherein the cell confluency reaches about 80-90%.

(2) Before transfection, changing the cell culture medium into serum-free DMEM medium;

(3) lipo2000 was used to transfect a total of 4. mu.g (pep3-Flag and NFATc3-GFP in a mass ratio of 2: 1) (NFATc 3-GFP: origene, cat # RG212523) of plasmid into cells

(4) Culturing in a 37 ℃ cell culture box for 4-6 h, and changing the culture solution into a fresh DMEM culture medium

(5) Adding 2 mu M (final concentration) CsA medicine into the positive control 24-48 h after transfection;

(6) culturing in a cell culture box at 37 ℃ for 1h, and adding 1 mu M (final concentration) of Ionomycin to stimulate for 2-6 h;

(7) taking out the cultured cell plate, removing the culture medium, adding precooled PBS, and washing for 3 times, each time for 5 min;

(8) adding precooled cell fixing solution (4% formaldehyde), and standing for 10min at room temperature;

(9) removing methanol and volatilizing, adding 1ml PBS and washing for 3 times, each time for 5 min;

(10) adding 300 μ l of 5mg/ml Hoechst 33258 nuclear staining solution, and incubating at room temperature for 10 min;

(11) discarding the staining solution, adding 1ml PBS to wash for 3 times, each time for 5 min;

(12) a drop of 5. mu.l of an anti-fluorescence quencher was dropped onto the slide, the cell slide was carefully mounted with the cell side down on the slide, and the slide was fixed with colorless nail polish and photographed by microscopic observation.

2. Results

When cell slides were observed using Immunofluorescence (IF) technique on transfected cells using ZEISS (ZEISS) LSM700 laser confocal microscopy, expression of pep3 short peptide was found to be effective in inhibiting Ionomycin (Ionomycin) activated NFAT nuclear entry behavior (fig. 7).

Example 7 inhibition of NFAT dephosphorylation by CN by pep3 short peptide-Western blotting

1. Cell processing

(1) The density of the HeLa cells in the fast growth phase of the inoculation is 5 multiplied by 10⁵Placing the cells in a 24-well plate with a cell slide, and culturing for 16-18 h (less than or equal to 24h) in a cell culture box at 37 ℃, wherein the cell recovery degree reaches about 80-90%.

(2) Before transfection, changing the cell culture medium into serum-free DMEM medium;

(3) a total of 4. mu.g (pep3-Flag and Myc-NFATc3 in a mass ratio of 2: 1) (Myc-NFATc 3: company: origene, RC200357L3) of plasmid was transfected into cells using Lipo 2000.

(4) Culturing in a 37 ℃ cell culture box for 4-6 h, and changing the culture solution into a fresh DMEM culture medium

(5) Adding 2 mu M (final concentration) CsA medicine into the positive control 24-48 h after transfection;

(6) culturing in a cell culture box at 37 ℃ for 1h, and adding 1 mu M (final concentration) of Ionomycin to stimulate for 2-6 h;

(7) taking out the cultured cell plate, removing the culture medium, and adding precooled PBS for washing and cleaning;

(8) discarding PBS, adding pancreatin, digesting for about 1min, blowing and beating cells into an EP tube, and centrifuging at 4 ℃ for 1000rpm for 5 min;

(9) discarding the supernatant, adding precooled PBS to wash and suspend, transferring to a new EP tube, and centrifuging at 4 ℃ for 1000rpm for 5 min;

(10) discarding the supernatant, adding 200 μ l RIPA, mixing, suspending, and standing in ice bath for 10 min;

(11) breaking cells by ultrasonic for 20s at 400W, centrifuging at 4 ℃ by 13000rpm by 10min, and taking out protein supernatant to a new centrifuge tube; (12) adding 5 times protein loading Buffer into the protein supernatant), boiling in water bath for 5min, and storing in a refrigerator at-20 deg.C.

2. Western blot

Same as example 2, step 7.

3. Results

Protein immunoblotting (Western Blot) of cell lysates revealed that intracellular expressed pep3 short peptide was effective in inhibiting the dephosphorylation ability of CN on NFAT (FIG. 8).

Example 8 Effect of pep3 short peptides on transcription levels of NFAT downstream factors-real-time fluorescent quantitative PCR

1. Cell processing

(1) The density of the HeLa cells in the fast growth phase of the inoculation is 5 multiplied by 10⁵Placing the cells in a 24-well plate with a cell slide, and culturing for 16-18 h (less than or equal to 24h) in a cell culture box at 37 ℃, wherein the cell recovery degree reaches about 80-90%.

(2) Each dish of cells was changed to serum-free DMEM medium before transfection.

(3) A total of 4. mu.g of pep3-Flag plasmid was transfected into cells using Lipo 2000.

(4) And (3) culturing the cells in a 37 ℃ cell culture box for 4-6 h, and then changing the culture solution into a fresh DMEM culture medium.

(5) And adding 2 mu M (final concentration) CsA drug into the positive control 24-48 h after transfection.

(6) After culturing in a 37 ℃ cell culture box for 1h, 1 mu M (final concentration) of Ionomycin is added for stimulation for 2-6 h.

2. Extraction of RNA

Placing the pore plate after collecting the culture solution on ice, and cleaning the culture medium; adding 1ml of Trizol into each hole, and standing for 3min on ice; sucking Trizol to blow and beat the resuspended cells, transferring the mixed solution to a 1.5ml centrifugal tube at the inlet, adding 1/5 volumes (200 mu l) of chloroform, swirling for 15s, and standing for 10min on ice; centrifuging at 4 deg.C for 12000g (13000rpm) for 18 min; sucking the upper aqueous phase into a new 1.5ml inlet centrifuge tube, adding 1/3 volumes of isopropanol, turning upside down and mixing uniformly, and standing on ice for 10 min; centrifuging at 4 deg.C for 12000g (13000rpm) for 12 min; discard the supernatant and directly reverse-buckle on clean filter paper, add 500. mu.l of 75% ethanol (from DEPC H)₂O preparation) blow-up RNA chip (indicating precipitated RNA in chip form) andrepeat 2 times; centrifuging at 4 deg.C × 12000g (13000rpm) × 5min, and repeating for 2 times; discarding ethanol, directly turning over on clean filter paper, covering with a cover, and blow-drying in a super clean bench (fume hood) for 5 min; add 10. mu.l DEPC H₂Dissolving RNA in O, measuring the concentration, subpackaging according to the dosage each time, and storing at-70 ℃.

3. Real-time fluorescent quantitative PCR

Using Takara (PrimeScript)^TMII Reverse Transcriptase Kit) Reverse transcribes the RNA to cDNA,using Takara (One Step PrimeScript)^TMRT-PCR Kit) to detect the expression of downstream genes IL-2 and TNF-alpha started by NFAT.

Wherein, the primer sequence for detecting the IL-2 gene is as follows:

IL-2-F：TGTCACAAACAGTGCACCTACTTC；

IL-2-R：TGTGGCCTTCTTGGGCATGT。

the primer sequences for detecting the TNF-alpha gene are as follows:

TNF-α-F：TCCTTCAGACACCCTCAACC；

TNF-α-R：AGGCCCCAGTTTGAATTCTT。

the internal reference is beta-actin gene, and the detection primers are as follows:

β-actin-F：GTGACAGCAGTCGGTTGGAG；

β-actin-R：AGTGGGGTGGCTTTTAGGAT。

4. results

The transcription level of mRNA was detected by fluorescent Real-time Quantitative PCR (Quantitative Real-time PCR) on transfected cells, and pep3 was found to effectively inhibit the expression of downstream genes IL-2 and TNF-alpha initiated by NFAT (FIG. 9, FIG. 10).

Examples 9, 11 ability of R-pep3 to enter cells-flow cytometry

1. Preparation of fluorescent membrane-penetrating short peptide

FITC-11R-pep3 was synthesized by Zhongke Asia photo Bio Inc. FITC-11R-pep3 is 11R-pep3(SEQ ID No.5) covalently linked at the N-terminus to a FITC (fluorescein isothiocyanate) fluorescent tag.

2. Incubation of membrane-penetrating short peptides

Collecting the suspension of the cultured Jurkat cells into a 15ml centrifuge tube, and centrifuging at 800rpm multiplied by 5 min; discarding the supernatant, adding 1ml RPMI-1640 culture medium (containing 10% FBS), gently sucking, mixing, counting with a hemocytometer at 5 × 10⁵Spreading the concentration of each/ml in a 6-well plate; placing the mixture in a constant temperature incubator at 37 ℃ for overnight culture, and dissolving the fluorescent transmembrane short peptide FITC-11R-pep3 in DMSO to prepare a mother solution with the concentration of 1 mM; adding 1-10 mu M FITC-11R-pep3 into a culture dish, and incubating for 30 min-2 h at 37 ℃; the cells were harvested and resuspended in 1ml PBSWashing cells, and centrifuging at 800rpm × 5 min; discarding the supernatant, adding 100 μ l PBS to resuspend the cells, sucking part of the cell fluid, and placing the cell fluid under a ZEISS upright fluorescence microscope for observation and photographing;

3. flow Cytometry (FC)

Adding 500 mul PBS into the washed cells for resuspension, and filtering by using a flow cytometer test tube to obtain single cell suspension; the ACEA flow cytometry system performs collection counting.

4. Results

After incubating the fluorescent transmembrane-spanning short peptide FITC-11R-pep3 into Jurkat cells in exponential growth phase and counting the fluorescent cells by using a flow cytometer, the modified 11R-pep3 was found to be able to enter the cells, and the number of the fluorescent cells and the short peptide had a dose-dependent effect (FIG. 11).

Example 10, 11 inhibition of ionomycin-activated endogenous NFAT dephosphorylation by R-pep 3-Western immunoblotting

1. Cell processing

(1) Collecting the suspension of the cultured Jurkat cells into a 15ml centrifuge tube, and centrifuging at 800rpm multiplied by 5 min;

(2) discarding the supernatant, adding 1ml RPMI-1640 culture medium (containing 10% FBS), gently sucking, mixing, counting with a hemocytometer at 5 × 10⁵Spreading the concentration of each/ml in a 6-well plate;

(3) placing the mixture in a constant temperature incubator at 37 ℃ for overnight culture, and dissolving the fluorescent transmembrane short peptide FITC-11R-pep3 in DMSO to prepare a mother solution with the concentration of 1 mM;

(4) adding FITC-11R-pep3 at 1 μ M and 5 μ M to the culture dish, adding cyclosporin A (CsA) at 1 μ M to the positive control group, and incubating at 37 deg.C for 1 h; then adding Ionomycin (Ionomycin) to 1 mu M, and incubating for 4h at 37 ℃;

(5) collecting cells, adding PBS to resuspend and wash the cells, and centrifuging at 800rpm multiplied by 5 min;

(6) discarding the supernatant, adding 200 μ l RIPA, mixing, suspending, and standing in ice bath for 10 min;

(7) breaking cells by ultrasonic for 20s at 400W, centrifuging at 4 ℃ by 13000rpm by 10min, and taking out protein supernatant to a new centrifuge tube;

(8) adding 5 times protein loading Buffer into the protein supernatant, boiling in water bath for 5min, and storing in a refrigerator at-20 deg.C.

2. Western blot

Reference is made to example 2, step 7.

3. Results

Cell lysates incubated with the short peptides were examined and western blotting experiments revealed that 11R-pep3 had a significant inhibitory effect on ionomycin-activated endogenous NFAT dephosphorylation (fig. 12).

Examples 11, 11 Effect of R-pep3 incubation on the expression of IL-2 Gene and protein, a downstream factor of NFAT-real-time fluorescent quantitation PCR and ELISA

1. Cell processing

(1) Collecting the suspension of the cultured Jurkat cells into a 15ml centrifuge tube, and centrifuging at 800rpm multiplied by 5 min;

(2) discarding the supernatant, adding 1ml RPMI-1640 culture medium (containing 10% FBS), gently sucking, mixing, counting with a hemocytometer at 5 × 10⁵Spreading the concentration of each/ml in a 6-well plate;

(3) placing the mixture in a constant temperature incubator at 37 ℃ for overnight culture, and dissolving the fluorescent transmembrane short peptide FITC-11R-pep3 in DMSO to prepare a mother solution with the concentration of 1 mM;

(4) adding FITC-11R-pep3 at 1 μ M and 5 μ M to the culture dish, adding cyclosporin A (CsA) at 1 μ M to the positive control group, and incubating at 37 deg.C for 1 h; then Ionomycin (Ionomycin) was added to 1. mu.M and incubated at 37 ℃ for 4 h.

2. Real-time fluorescent quantitative PCR

Reference is made to example 8.

3、ELISA

Taking out the IL-2 antibody coupled ELISA special-purpose pore plate, recovering to room temperature, and not returning to 4 ℃; collecting the cultured Jurkat cell suspension (the cells processed in the step 1) into a 15ml centrifuge tube, and centrifuging at 800rpm for 5 min; adding 100 μ l of IL-2 standard dilution into blank control wells, adding 100 μ l of cell supernatants of different groups into experimental group wells, sealing the use wells with sealing plate adhesive paper, and incubating at 37 deg.C for 90 min; washing the plate for 5 times; adding 100 μ l biotin antibody diluent into blank control group, adding 100 μ l biotinylated antibody working solution into experimental group, sealing the use hole with new sealing plate adhesive paper, and incubating at 37 deg.C for 60 min; washing plate 5Secondly; adding 100 μ l enzyme conjugate diluent into blank control group, adding 100 μ l enzyme conjugate working solution into experimental group, sealing the use hole with new sealing plate adhesive paper, and incubating at × 37 deg.C for 30min in dark; washing the plate for 5 times; adding 100 μ l of chromogenic substrate (TMB) to each group, and incubating for 15min at × 37 ℃ in the dark; adding 100 μ l stop solution into each group, mixing, and immediately (less than or equal to 3min) measuring OD₄₅₀；

4. Results

Fluorescence real-time quantitative PCR and ELISA (enzyme linked immunosorbent assay) technology detect that the mRNA transcription level and the protein expression level of the IL-2 gene in the cells incubated with the short peptides are obviously reduced (FIG. 13, FIG. 14).

Example 12, 11 construction of mouse asthma model with immunosuppressive function of R-pep3 on Individual at animal level

1. Grouping of mice

SPF-grade 6-8 week-old female BALB/C mice are randomly divided into 4 groups, and each group comprises 5 mice; a normal control group (MOCK group), OVA group, FITC-11R-pep3 asthma group, FITC-11R-PVIVIT asthma group, and the first day of the inclusion cage were set.

2. Molding die

The mice are molded seven days after entering the cage, and the treatment is as follows:

sham group (sham group): injecting 200 μ l normal saline (containing 100 μ l aluminum hydroxide gel) into abdominal cavity on 7 days and 14 days, and dripping 50 μ l normal saline into nasal cavity for administration and excitation before excitation on 21-24 days;

OVA group (ovalbumin group): intraperitoneal injection of 200 μ l of chicken Ovalbumin (OVA) sensitizing agent (containing 10 μ g of OVA dissolved in normal saline and 100 μ l of aluminum hydroxide gel) on 7 th and 14 th days, nasal administration of 50 μ l of normal saline before challenge on 21 st to 24 th days, and challenge administration of 50 μ l of 1% OVA solution (dissolved in normal saline,% represents g/100 ml);

FITC-11R-pep3 group/FITC-11R-PVIVIT group: on day 7 andday 14, 200. mu.l of OVA sensitizing agent (containing 10. mu.g of OVA dissolved in physiological saline and 100. mu.l of aluminum hydroxide gel) was intraperitoneally injected, and 50. mu.l of 1% OVA solution (containing 200. mu.g of active polypeptide dissolved in physiological saline) was administered by nasal drip before challenge on day 21 to day 24, and challenge administration was performed.

FITC-11R-pep3 and FITC-11R-PVIVIT used in this example were both synthesized by Hitachi Adam Biotech.

3. Making HE stained section of lung

After the molding is finished, fixing the right lung of the mouse in 4% formaldehyde for 24-72 h; after dehydration, paraffin embedding is carried out, and after the embedded tissue block becomes hard, slicing is carried out. Appropriate sections were mounted on a patch and dried and then HE stained: paraffin is removed by xylene, the mixture is gradually cleaned by alcohol and distilled water, and hematoxylin staining solution and eosin staining solution are used for staining successively. And (5) dehydrating the dyed slices by using alcohol again, and sealing the slices after transparent treatment.

4. Results

A mouse asthma model is constructed by using chicken Ovalbumin (OVA) sensitization, and the short peptide is administrated by nasal drip before OVA excitation, and the result shows that 11R-pep3 can effectively intervene the generation of mouse asthma and play an immunosuppressive function at the animal level (figure 15).

Sequence listing

<110> university of Beijing teachers

<120> short peptide inhibitor targeting calmodulin phosphatase and T cell activation nuclear factor as substrate thereof and application thereof

<130> GNCLN191309

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> PRT

<213> Artificial sequence

<400> 1

Glu Lys Tyr Glu Leu His Ala Ala Thr Asp Thr Thr Pro Ser Val Val

1 5 10 15

Val His Val Cys Glu Ser

20

<210> 2

<211> 15

<212> PRT

<213> Artificial sequence

<400> 2

Asp Gln Tyr Leu Ala Val Pro Gln His Pro Tyr Gln Trp Ala Lys

1 5 10 15

<210> 3

<211> 16

<212> PRT

<213> Artificial sequence

<400> 3

Gly Cys Glu Asp Asn Val Tyr Glu Lys Leu Pro Glu Gln Asn Ser Asn

1 5 10 15

<210> 4

<211> 49

<212> PRT

<213> Artificial sequence

<400> 4

Pro Gly Glu Lys Tyr Glu Leu His Ala Ala Thr Asp Thr Thr Pro Ser

1 5 10 15

Val Val Val His Gly Cys Glu Asp Asn Val Tyr Glu Lys Leu Pro Glu

20 25 30

Gln Asn Ser Asn Tyr Leu Ala Val Pro Gln His Pro Tyr Gln Trp Ala

35 40 45

Lys

<210> 5

<211> 63

<212> PRT

<213> Artificial sequence

<400> 5

Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Gly Gly Pro Gly

1 5 10 15

Glu Lys Tyr Glu Leu His Ala Ala Thr Asp Thr Thr Pro Ser Val Val

20 25 30

Val His Gly Cys Glu Asp Asn Val Tyr Glu Lys Leu Pro Glu Gln Asn

35 40 45

Ser Asn Tyr Leu Ala Val Pro Gln His Pro Tyr Gln Trp Ala Lys

50 55 60

<210> 6

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> the amino acid is modified by phosphorylation

<400> 6

Asp Leu Asp Val Pro Ile Pro Gly Arg Phe Asp Arg Arg Val Ser Val

1 5 10 15

Ala Ala Glu

<210> 7

<211> 66

<212> DNA

<213> Artificial sequence

<400> 7

gaaaagtatg aattgcacgc agcgactgac accactccca gcgtggtggt ccatgtatgt 60

gagagt 66

<210> 8

<211> 45

<212> DNA

<213> Artificial sequence

<400> 8

gatcagtact tggccgtacc acagcatccg tatcaatggg ctaag 45

<210> 9

<211> 48

<212> DNA

<213> Artificial sequence

<400> 9

ggctgtgaag acaatgtcta tgaaaaactt cctgaacaga attcaaac 48

<210> 10

<211> 147

<212> DNA

<213> Artificial sequence

<400> 10

ccaggagaga agtatgaact gcatgcagcg acagacacca ctcccagtgt ggtggtccac 60

ggctgtgaag acaatgtcta tgaaaaactt cctgaacaga attcaaacta cctggcggtg 120

ccgcagcacc cctaccagtg ggcgaag 147

Claims (16)

1. A fusion polypeptide which is (a1) or (a2) as follows:

(A1) a fusion polypeptide 1 formed by connecting EV motif of calmodulin phosphatase regulatory factor 1 and LxVP motif of T cell activating nuclear factor 1 through A238L connecting peptide;

(A2) fusion polypeptide 2, which is obtained after connecting a membrane-penetrating peptide to the amino terminal and/or the carboxyl terminal of the fusion polypeptide 1 of (a 1);

the EV motif of the calmodulin phosphatase regulatory factor 1 is a polypeptide shown in SEQ ID No. 1;

the LxVP motif of the T cell activated nuclear factor 1 is polypeptide shown as SEQ ID No. 2;

the A238L connecting peptide is polypeptide shown in SEQ ID No. 3.

2. The fusion polypeptide of claim 1, wherein: the cell-penetrating peptide is a polypeptide shown in 1 st to 14 th positions of SEQ ID No. 5.

3. The fusion polypeptide of claim 1, wherein: the fusion polypeptide 1 is a polypeptide shown as SEQ ID No. 4.

4. The fusion polypeptide of claim 2, wherein: the fusion polypeptide 2 is a polypeptide shown as SEQ ID No. 5.

5. A fusion polypeptide modifier, characterized in that: the modified fused polypeptide is obtained by carrying out fluorescence labeling on the amino terminal and/or the carboxyl terminal of the fused polypeptide of any one of claims 1 to 4.

6. A nucleic acid molecule encoding the fusion polypeptide of any one of claims 1-4.

7. The nucleic acid molecule of claim 6, wherein: in the nucleic acid molecule, the nucleic acid molecule encoding the EV motif of calmodulin phosphatase regulatory factor 1 is a DNA molecule shown in SEQ ID No. 7.

8. The nucleic acid molecule of claim 6, wherein: in the nucleic acid molecule, the nucleic acid molecule encoding the LxVP motif of the T cell activating nuclear factor 1 is a DNA molecule shown in SEQ ID No. 8.

9. The nucleic acid molecule of claim 6, wherein: in the nucleic acid molecule, the nucleic acid molecule encoding the A238L connecting peptide is a DNA molecule shown in SEQ ID No. 9.

10. The nucleic acid molecule of any one of claims 6-9, wherein: the nucleic acid molecule for coding the fusion polypeptide 1 is a DNA molecule shown in SEQ ID No. 10.

11. A recombinant vector, expression cassette, recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of any one of claims 6 to 10.

12. Any of the following applications:

(C1) use of a modified fusion polypeptide according to any one of claims 1 to 4 or a modified fusion polypeptide according to claim 5 or a nucleic acid molecule according to any one of claims 6 to 10 or a recombinant vector, expression cassette, recombinant bacterium or transgenic cell line according to claim 11 for the preparation of an immunosuppressant;

(C2) use of a modified fusion polypeptide according to any one of claims 1 to 4 or a modified fusion polypeptide according to claim 5 or a nucleic acid molecule according to any one of claims 6 to 10 or a recombinant vector, expression cassette, recombinant bacterium or transgenic cell line according to claim 11 for the preparation of a product for the prevention and/or treatment of hypersensitivity disorders;

(C3) use of a modified fusion polypeptide according to any one of claims 1 to 4 or a modified fusion polypeptide according to claim 5 or a nucleic acid molecule according to any one of claims 6 to 10 or a recombinant vector, expression cassette, recombinant bacterium or transgenic cell line according to claim 11 for the preparation of a product for the prevention and/or treatment of an autoimmune disease;

(C4) use of a modified fusion polypeptide according to any one of claims 1 to 4 or a modified fusion polypeptide according to claim 5 or a nucleic acid molecule according to any one of claims 6 to 10 or a recombinant vector, expression cassette, recombinant bacterium or transgenic cell line according to claim 11 for the preparation of a product for the prevention and/or treatment of an allergic reaction;

(C5) use of a modified fusion polypeptide according to any one of claims 1 to 4 or a modified fusion polypeptide according to claim 5 or a nucleic acid molecule according to any one of claims 6 to 10 or a recombinant vector, expression cassette, recombinant bacterium or transgenic cell line according to claim 11 for the manufacture of a product for the prevention and/or treatment of asthma;

(C6) use of a modified fusion polypeptide according to any one of claims 1 to 4 or a modified fusion polypeptide according to claim 5 or a nucleic acid molecule according to any one of claims 6 to 10 or a recombinant vector, expression cassette, recombinant bacterium or transgenic cell line according to claim 11 for the manufacture of a product for inhibiting immune rejection in an organ transplant.

13. Use of a modified fusion polypeptide according to any one of claims 1 to 4 or a modified fusion polypeptide according to claim 5 or a nucleic acid molecule according to any one of claims 6 to 10 or a recombinant vector, expression cassette, recombinant bacterium or transgenic cell line according to claim 11 for the manufacture of a product having at least one of the following functions:

(D1) binding to calmodulin phosphatase;

(D2) inhibiting dephosphorylation of a substrate by calcineurin;

(D3) inhibiting the nuclear entry behavior of ionomycin-activated T cells activating nuclear factors;

(D4) inhibiting dephosphorylation of a T cell activating nuclear factor by calcineurin phosphatase;

(D5) inhibiting the expression of downstream genes initiated by T cell activating nuclear factors.

14. Use according to claim 13, characterized in that: (D2) wherein the substrate is a RII peptide.

15. Use according to claim 14, characterized in that: the RII peptide is a polypeptide shown as SEQ ID No. 6.

16. Use according to claim 13, characterized in that: (D5) wherein the downstream gene is IL-2 and/or TNF-alpha.

Images